Extensively expanded murine‐induced hepatic stem cells maintain high‐efficient hepatic differentiation potential for repopulation of injured livers

2020 ◽  
Vol 40 (9) ◽  
pp. 2293-2304
Author(s):  
Bing Yu ◽  
Hengyu Li ◽  
Jie Chen ◽  
Zhiying He ◽  
Haixiang Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hepatic Stem Cells ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
High Efficient

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

Clonogenic Colony-Forming Ability of Flow Cytometrically Isolated Hepatic Progenitor Cells in the Murine Fetal Liver

2000 ◽  
Vol 9 (5) ◽  
pp. 697-700 ◽  
Author(s):  
Hideki Taniguchi ◽  
Reika Kondo ◽  
Atsushi Suzuki ◽  
Yun-Wen Zheng ◽  
Yasutsugu Takada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Therapeutic Potential ◽  
Fetal Liver ◽  
Cell Isolation ◽  
Hepatic Stem Cells ◽  
Differentiation Potential ◽  
Isolation Technique ◽  
A Cell ◽  
Hepatocyte Markers ◽  
Facs Sorting

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45–TER119– cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45–TER119– cells in the murine fetal liver.

Cell Heterogeneity, Rather Than Cell Solvents Affects the Behavior of Mesenchymal Stem Cells in Vitro and Vivo

2020 ◽  
Author(s):  
Yong-Hong Wang ◽  
Ya-Chao Tao ◽  
Dong-Bo Wu ◽  
Meng-Lan Wang ◽  
Hong Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Survival Rates ◽  
Phosphate Buffer Solution ◽  
Human Umbilical Cord ◽  
Cell Heterogeneity ◽  
Hepatic Differentiation ◽  
Buffer Solution ◽  
Differentiation Potential

Abstract Background: Mesenchymal stem cells (MSCs) have been approved to treat various diseases, but they have to be expanded in vitro to gain sufficient cell doses. During the process of expansion, some obstacles remain to be addressed before MSCs translation to clinic. The purpose of this study was to investigate the effects of cell solvents and cell heterogeneity on the behavior of MSCs in vitro and vivo.Methods: Human umbilical cord MSCs (UC-MSCs) were dissolved in three different solvents: phosphate buffer solution (PBS), normal saline (NS) and dulbecco's modified eagle medium (DMEM). Their ultrastructure, viability and safety were explored and compared. MSCs from other two separate donors were grouped based on their mean diameters. The ultrastructure, proliferative and hepatic differentiation potential, senescent cell ratio and safety of the two UC-MSC aggregates were investigated and compared. The reason for mice death after UC-MSCs injection was further investigated.Results: The apoptosis rates, ultrastructure analysis and survival rates of mice among UC-MSCs in DMEM, NS and PBS were similar, and no significant differences were observed. The diameters of UC-MSCs of different sizes were measured. Cells with diameter of 15.58±3.813 μm were renamed as larger UC-MSC aggregates and cells with diameter of 19.14±4.885 μm were smaller aggregates. The mean diameter of larger MSC aggregates was significantly longer than that of smaller aggregates (p<0.01). Smaller MSCs had more potent proliferation potential and higher nucleus/ cytoplasm ratio than large ones. The number of cells positive for senescence-associated β-galactosidase staining was higher in larger UC-MSC aggregates. The survival rates of mice receiving 1×106 or 2×106 smaller MSCs were 100%, both higher than that receiving larger UC-MSCs sharing same amount. Meanwhile, the reason for mice death was explored and it revealed that larger UC-MSC aggregates were accumulated and evident in the pulmonary capillary lumen in dead mice.Conclusion: Solvents showed no significant effects on cell behavior, whereas, heterogeneity is quite prevalent in MSCs populations and may limit cell application, but it is easily overlooked. Hence it is necessary to establish a more precise standardization for culture-expanded MSCs and to improve MSCs manufacturing strategies.

scholarly journals Cell heterogeneity, rather than the cell storage solution, affects the behavior of mesenchymal stem cells in vitro and in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yong-Hong Wang ◽  
Ya-Chao Tao ◽  
Dong-Bo Wu ◽  
Meng-Lan Wang ◽  
Hong Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Survival Rates ◽  
Phosphate Buffer Solution ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
Normal Ranges ◽  
Storage Solutions

Abstract Background Mesenchymal stem cells (MSCs) have to be expanded in vitro to reach a sufficient cell dose for the treatment of various diseases. During the process of expansion, some obstacles remain to be overcome. The purpose of this study was to investigate the effects of storage solutions and heterogeneity on the behavior of MSCs in vitro and in vivo. Methods Umbilical cord MSCs (UC-MSCs) of similar sizes within normal ranges were suspended in three different storage solutions, phosphate buffer solution, normal saline, and Dulbecco’s modified Eagle medium. Then, the ultrastructure, viability, and safety of these cells were compared. Other two UC-MSC populations of different sizes were categorized based on their mean diameters. The ultrastructure, proliferation, immunosuppression, hepatic differentiation potential, and number of senescent cells were investigated and compared. The survival rates of mice after the infusion of UC-MSCs of different sizes were compared. Results For UC-MSCs suspended in different storage solutions, the cell apoptosis rates, ultrastructure, and survival rates of mice were similar, and no differences were observed. Cells with a diameter of 19.14 ± 4.89 μm were categorized as the larger UC-MSC population, and cells with a diameter of 15.58 ± 3.81 μm were categorized as the smaller population. The mean diameter of the larger UC-MSC population was significantly larger than that of the smaller UC-MSC population (p < 0.01). Smaller UC-MSCs had more powerful proliferation and immunosuppressive potential and a higher nucleus-cytoplasm ratio than those of large UC-MSCs. The number of cells positive for β-galactosidase staining was higher in the larger UC-MSC population than in the smaller UC-MSC population. The survival rates of mice receiving 1 × 106 or 2 × 106 smaller UC-MSCs were 100%, both of which were higher than those of mice receiving the same amounts of larger UC-MSCs (p < 0.01). The cause of mouse death was explored and it was found that some larger UC-MSCs accumulated in the pulmonary capillary in dead mice. Conclusion Different storage solutions showed no significant effects on cell behavior, whereas heterogeneity was quite prevalent in MSC populations and might limit cells application. Hence, it is necessary to establish a more precise standardization for culture-expanded MSCs.

scholarly journals GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells

2015 ◽  
Vol 35 (6) ◽  
pp. 2299-2308 ◽  
Author(s):  
Jiong Yu ◽  
Xiaoru Su ◽  
Chengxing Zhu ◽  
Qiaoling Pan ◽  
Jinfeng Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Human Placenta ◽  
Fluorescent Protein ◽  
Gene Transduction ◽  
Hepatic Differentiation ◽  
Differentiation Potential ◽  
Gfp Gene ◽  
Cell Based Therapy

Background: Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. Methods: The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP+ hPMSCs. GFP+ hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP+ hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. Results: GFP+ hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP+ hPMSCs showed typical hepatic phenotypes. Conclusions: GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP+ hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy.

scholarly journals Physiological Hypoxia Enhances Stemness Preservation, Proliferation, and Bidifferentiation of Induced Hepatic Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaosong Zhi ◽  
Jun Xiong ◽  
Mengchao Wang ◽  
Hongxia Zhang ◽  
Gang Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Hepatic Stem Cells ◽  
Hepatic Differentiation ◽  
Proliferation And Differentiation ◽  
Proliferation Ability ◽  
Cell Niche ◽  
Promising Perspective

Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their self-renewal and bipotential differentiation properties. However, the efficiency of bidifferentiation and repopulation efficiency of iHepSCs is relatively low. Recent evidence shows that physiological hypoxia, a vital factor within stem cell “niche” microenvironment, plays key roles in regulating tissue stem cell biological behaviors including proliferation and differentiation. In this study, we found that physiological hypoxia (10% O2) enhanced the stemness properties and promoted the proliferation ability of iHepSCs by accelerating G1/S transition via p53-p21 signaling pathway. In addition, short-term hypoxia preconditioning improved the efficiency of hepatic differentiation of iHepSCs, and long-term hypoxia promoted cholangiocytic differentiation but inhibited hepatic differentiation of iHepSCs. These results demonstrated the potential effects of hypoxia on stemness preservation, proliferation, and bidifferentiation of iHepSCs and promising perspective to explore appropriate culture conditions for therapeutic stem cells.

Sign in / Sign up

Export Citation Format

Share Document